Custom Antibody Production

Should I generate a polyclonal or a monoclonal antibody?

Choosing between producing a polyclonal or a monoclonal antibody depends on multiple factors such as timeline, budget, or purpose. Polyclonal production is usually faster than monoclonal production by 1-2 months. The budget to generate a monoclonal is at least 4 times higher than for a polyclonal. A monoclonal antibody allows for unlimited production with sustained specificity whereas a polyclonal antibody is limited to the volume of blood collected from one animal, using another animal will allow for specificity variation. By definition a monoclonal is specific of a unique epitope whereas a polyclonal antibody recognizes multiple epitopes within a peptide or a protein, making the two types of antibodies suitable for different applications.

What type of antibody production services does Abbiotec offer?

Abbiotec is focusing on making polyclonal antibodies in rabbits. Species like goat, rat or mouse can be used on special request. Monoclonal antibody production in mouse and rat can be offered upon project approval. Contact our Technical Support Dept for these special requests.

Polyclonal antibody production requires 3-4 months depending on antigen and length of immunization procedure. Usually, the process is faster when using a protein than for a peptide. If the animal does not respond to the immunization, more boosts are planned before blood collection. Monoclonal antibody production requires 5-6 months.

What kind of antigens do you use to make antibodies?

Abbiotec’s protocols use small molecules, peptides, native and recombinant proteins to make antibodies. When the antigen is smaller than 10 kDa, the antigen is conjugated to a carrier protein such as KLH to make the antigen immunogenic. For peptide antigens , we recommend adding a cysteine residue at the C-terminus end of the peptide sequence to allow for efficient conjugation using the free sulhydryl chemical group.

Can Abbiotec help me design a good peptide sequence for my antigen?

Abbiotec technical staff is highly experienced in antibody development and epitope design. Whereas most companies rely on bioinformatic algorithms, Abbiotec staff is able to take into account the protein surface available for antibody binding by using a 3D protein design software. Ultimately, the peptide candidates are reviewed by a qualified Ph.D. scientist before the final recommendations are sent to the client. This service is free of charge when Abbiotec synthesizes the peptide. Contact our Technical Support Dept to send your protein sequence or corresponding protein accession number.

Do tags interfere with antibody production?

Abbiotec does not recommend fusion proteins when producing an antibody. Large tag proteins such as GST and MBP are highly immunogenic and therefore dilute the population of antibodies made against the fused fragment. Abbiotec recommends cleaving the fusion off before immunization, especially for small fragment fused to large tags. Other tags like 6xHis, Myc, or FLAG can be used without biasing the immune response against the fused fragment.

How much antigen is required for antibody development?

For animal immunization, 10 mg of a peptide is required. An additional 5 mg is needed when antigen-affinity purification is performed. The peptide should be supplied in a solid format free of toxic additives such as PMSF or sodium azide. When a protein is used as antigen, 5 mg is sufficient. When the protein is in solution, the concentration should be at 1 mg/ml in a buffer solution free of detergents. When the protein is insoluble, using an aggregate is the best format instead of a solubilization buffer containing denaturing agents.

How should I send the antigen to Abbiotec?

Abbiotec recommends using a major carrier company to send your sample for next day delivery. Use a 15-ml conical tube or insert a smaller vial into a 50-ml conical tube to avoid loss of sample due to vial breakage. If the sample is temperature sensitive, use a styrofoam box with ice packs.

Can you guarantee that my antibody will work for me?

Abbiotec offers you the best recommendations and antibody production experience to minimize the chances to fail at making the requested antibody. There is no guarantee that the antibody that Abbiotec produces for you will work for your application. Some projects are at higher risks than others and Abbiotec will inform you of the chance of failure at project initiation. Abbiotec still guarantees that the antibody generated using an antigen synthesized by Abbiotec will recognize the antigen in a direct ELISA.

How should antibodies from my project be stored?

Antibodies that went through a purification process are supplied in PBS buffer without preservatives. Anti-sera collected from blood are supplied crude with 0.1% sodium azide. Purified antibodies and anti-sera are stable at -80°C for 1 year. Multiple freeze-thaw cycles degrade antibodies, therefore we recommend aliquoting small amounts of antibodies for short term storage at -20°C up to a month or 4°C up to 3 days.

When will I be billed for a custom antibody project?

A first invoice corresponding to 40% of full amount is sent at project initiation and a second invoice corresponding to remaining balance including S&H fees is sent with final shipment of products.

Although most peptides can be shipped out within 2-3 weeks, actual processing time is dependent on the peptide sequence, length, purity and modifications. Please consult with our Technical Support Dept for an estimated timeline of the peptide synthesis process. Estimated delivery times are stated on all quotations.

What type of chemistry does Abbiotec use to synthesize peptides?

Peptides from Abbiotec are made using F-moc solid phase method. Automated or manual peptide synthesis is performed where appropriate.

How are Abbiotec peptides supplied?

Peptides are lyophilized in a solid powder format under an inert gas and as trifluoroacetate salt.

How do I store peptides?

The lyophilized peptides should be stored at -20°C to maintain maximum stability over time. Before reconstitution, equilibrate the vial at room temperature to avoid absorption of water. Storage at 4°C is acceptable for working stock solution. We recommend making fresh stock solution for critical assays since slow degradation such as oxidation occurs once the peptide is reconstituted in solution.

How do I dissolve a lyophilized peptide?

Solubility of a peptide depends on the amino acid sequence. If no recommendation is provided with the product, use sterile water first to reconstitute a sample of the peptide at 1-10 mg/ml. Sonication of the solution helps dissolving aggregates. If the peptide is not 100% recovered, add 0.1 M ammonium hydroxide drop-wise for acidic peptides, or add 0.1 M acetic acid drop-wise for basic peptides. Acidity index of a peptide can be estimated by counting acidic residues (Glu, Asp and free C-terminus) versus basic residues (Lys, Arg, His and free N-terminus). Do not add salt buffers before full reconstitution of the peptide in solution. Organic solvents such as DMSO, DMF, DMA, isopropanol or ethanol, can be used for peptides poorly soluble in water. We recommend using 100% solvent first and dilute with water as appropriate.

What peptide purity level do I need for my application?

Abbiotec peptides are purified by reverse phase HPLC chromatography. A crude peptide corresponds to a purity of 70% with salt and solvent contaminants (sodium acetate and trifluoroacetic acid) whereas a HPLC-purified peptide has a purity of 90 to 98% free of contaminants. Peptides with purity >70% can be used for antibody production. For other applications, we recommend using a purity >90%, a purity >98% being reserved for specific assays and in vivo studies.